S-eluorourace



United States Patent ice Patented Aug. 6, 1957 S-FLUQRUURAQHL CharlesHeidelherger, Madison, Wis, and Robert Duschinslry, Essex Fells, N. 3.,

No Drawing. Application September 26, 1956, Serial No. 612,032;

12 Claims. (Cl. 26li--260) RA represents a lower alkylating agent;preferably a lower alkyl ester of an inorganic mineral acid, such asdiethyl sulfate, methyl bromide, ethyl iodide, and the like: in thepreferred case, R represents a lower alkyl radical and A represents ananionic portion of said mineral acid.

M and M each represents an alkali metal, for example potassium orsodium.

R and R each represents a lower alkyl radical.

R represents a radical selected from the group consisting of lower alkyland benzyl.

X represents a halo substituent selected from the group consisting ofchloro and brorno.

It will be understood that none of the lower alkyl radicals representedby the symbols R R R and R need be identical; but any or all of suchradicals can be identical alkyl radicals. Likewise, the alkali metalsrepresented by the symbols M and M need not be identical; but suchmetals can be identical.

FLOW SHEET-PROCESS II FCHQOO OR" (21) R 0 0 0 0 0 0 R M(0r M-O-RD] i 0 Ral FLOW SHEET-PROCESS III R-S-O G LE =l l H N I H FLOW SHEETPROCESS IVFLOW SHEET-PROCESS V PROCESS I As will be appreciated from the foregoingflow sheet for Process 1, a comprehensive embodiment of this aspect ofthe invention provides a process which comprises reacting alkali metalfluoroacetate (11) with lower alkylating agent (12), thereby producinglower alkyl fluoroacetate (13); subjecting the latter to Claisencondensation with lower alkyl formate (14) in the presence of alkalimetal-containing condensing agent (15), thereby producing alkali metalenolate of lower alkyl fluoromalonaldehydate (16); condensing the latterunder anhydrous conditions with a member (17) selected from the groupconsisting of S-(lower alkyl)-isothiourea and S-benzylisothiourea,thereby producing the corresponding member (18) of the group consistingof S-loWer alkyl ether of 2-thio-5-fiuorouracil and S-benzyl ether of 2-thio-S-fiuorouracil; and hydrolyzing said corresponding member, therebyproducing S-fluorouracil (19).

The first stage of the comprehensive embodiment of Process I referred toabove comprises reacting the alkali metal fluoroacetate (11) with alower alkylating agent (12). Preferably the sodium or potassium salt offluoroacetic acid is used as reactant (11); and preferably diethylsulfate or dimethyl sulfate or methylbromide or ethyl chloride or thelike is used as the lower alkylating agent (12). The reaction can beeffected, for example, by heating the reactants together untilcompletion of the reaction whereby the lower alkyl radical is exchangedfor the alkali metal. An inert solvent can be employed, if desired, butits use is not required. The product can be purified, if desired, byconventional means, for example by distillation.

The second stage of said comprehensive embodiment comprises reacting thelower alkyl fluoracetate (13) in a Claisen condensation with lower alkylformate (14), employing an alkali metal-containing condensing agent(15), such as an alkali metal itself, e. g. potassium or sodium, or analkali metal lower alkoxide, e. g. potassium ethoxide or sodiummethoxide. Preferably a potassium lower alkoxide, such as potassiumethoxide, is employed as condensing agent; the potassium enolate therebyproduced as product can be easily isolated in well crystallized form. Asuitable method of effecting this second stage of the overall processcomprises heating the reactants (13), (14) and (15) together, in ananhydrous inert solvent, such as diethyl ether or toluene, untilcompletion of the condensation. The product, represented by generalformula (16) in the flow sheet, can be styled alkali metal enolate oflower alkyl fluoromalonaldehydate; alternative nomenclatures are, alkalimetal salt of lower alkyl ester of fluoromalonaldehydic acid, or alkalimetal salt of lower alkyl ester of forrnylfluoroacetic acid.Particularly when potassium lower alkoxides are used as condensingagents, the product 16) need not be purified by recrystallization forfurther use in the process.

The third stage of the comprehensive embodiment of Process I shown inthe flow sheet comprises condensing the alkali metal enolate of loweralkyl fluorom'alonaldehydrate (16) with (17), i. e. S-(loweralkyl)isothiourea or S-benzylisothiourea, under anhydrous conditions.Preferably, the reaction is effected by heating the reactants togetherin an anhydrous inert solvent, such as methanol or ethanol, until thecondensation reaction has been completed. It is recommended to use thealkali metal enolate reactant (16) as soon as possible after it has beenprepared. It is preferred to use freshly prepared alkali metal enolatereactant (16), i. e. material which is not older than two (or at mostthree) days old; and preferably the alkali metal enolate should beusedas soon as it is prepared. The S-(lower alkyl)- isothiourea orS-benzylisothiourea is advantageously employed in the form of its acidaddition salt with a mineral acid, e. g. S-methylisothiouronium sulfateor S- ethylisothiouroniurn bromide or S-benzylisothiouronium chloride,in the presence of at least the equivalent amount of alkali necessary toneutralize the mineral acid. The product, S-lower alkyl (or S-benzyl)ether of 2-thio-5- fluorouracil (18), can be purified byconventionalmeans, e. g. by recrystallization from inert solvents.

The last stage of the comprehensive embodiment shown in the flow sheetfor Process I comprises hydrolyzing the S-lower alkyl (or S-benzyl)ether of 2-thio-5-fiuorouracil (18) thereby producing S-fluorouracil(19). The hydrolysis can be conveniently eifected by conventional means,for example by heating the reactant (18) with an aqueous solution of amineral acid, e. g. with concentrated aqueous hydrochloric acid. TheS-fluorouracil obtained as product can be purified by conventionalmeans, for example by recrystallization from inert solvents or bysublimation in vacuo.

One preferred embodiment of the overall Process I shown in the flowsheet comprises reacting sodium fluoroacetate with diethyl sulfate,thereby producing ethyl fluoroacetate; reacting the latter in a Claisencondensation with ethyl formate in thepresence of potassium ethoxide,thereby producing potassium enolate of ethyl fluoro- Inalonaldehydate;condensing freshly prepared potassium enolate of ethylfluoromalonaldehydate under anhydrous conditions withS-methylisothiourea, thereby producing S-methyl ether ofZ-thio-S-fiuorouracil; and hydrolyzing the latter, thereby producing-fluorouracil. Still another preferred embodiment of the overall ProcessI comprises the same operations in the same sequence; except thatS-ethylisothiourea is employed in lieu of S- methylisothiourea in thepenultimate step, S-ethyl ether of Z-thio-S-fluorouracil being therebyproduced in lieu of S-methyl ether of 2-thio-5-fiuorouracil.

PROCESS II As will be appreciated from the foregoing flow sheet forProcess II, a comprehensive embodiment of this second aspect of theinvention provides a process which cornprises subjecting lower alkylfluoroacetate (21) to Claisen condensation with di(lower alkyl) oxalate(22) in the presence of alkali metal-containing condensing agent (23),thereby producing alkali metal enolate of di(lower alkyl)fiuorooxalaeetate (24); condensing the latter with a member (25)selected from the group consisting of 8- (lower alkyl)-isothiourea andS-benzylisothiourea, thereby producing the corresponding member (26)selected from the group consisting of S-lower alkyl ether of 2-thio-S-fluoroorotic acid lower alkyl ester and S-benzyl ether ofZ-thio-S-fiuoroorotic acid lower alkyl ester; hydrolyzing saidcorresponding member, thereby producing 5-fiuoroorotic acid (27); anddecarboxylating the latter, thereby producing S-fluorouracil (28) Thefirst stage of the comprehensive embodiment of Process II referred toabove comprises reacting lower alkyl fluoroacetate in a Claisencondensation with di(lower alkyl) oxalate, in the presence of alkalimetal-containing condensing agent. Preferably, ethyl fluoroacetate isemployed with diethyl oxalate; or, alternatively, methyl fluoroacetatewith dimethyl oxalate. As the alkali metalcontaining condensing agent,preferably potassium ethoxide is employed; but, alternatively, potassiummethoxide or potassium can be employed. A suitable method of effectingthis first stage comprises heating the reactants (21), (22) and (23)together in an anhydrous inert solvent such as ethanol, toluene ordiethyl ether, until completion of the condensation. The product,represented by the general formula (24) in the flow sheet for ProcessII, can be styled alkali metal enolate of di(lower 6 alkyl)fluorooxalacetate; an alternative nomenclature is, alkali metal salt oflower alkyl ester of fluorooxalacetic acid. Particularly when ethylfluoroacetate is condensed with diethyl oxalate in the presence ofpotassium ethoxide, the resulting product, diethylpotassio-fluorooxalacetate, need not be purified before further reactionin Process II.

I The second stage of the comprehensive embodiment of Process 11referred to above comprises condensing alkali metal enolate of di(loweralkyl) fluorooxalacetate with S-(lower alkyl)isothiourea orS-benzylisothiourea. Advantageously, the S-(lower alkyl)isothiourea orS-benzylisothiourea is employed in the form of its acid addition saltwith a mineral acid, e. g. S-methylisothiouronium sulfate orS-ethylisothiouronium bromide or S-benzylisothiouronium chloride, in thepresence of at least the equivalent amount of alkali necesary toneutralize the mineral acid. The reaction is preferably eifected byheating the reactants together in an anhydrous inert solvent, such asmethanol or ethanol, until the condensation reaction has been completed.It is recommended to use freshly pre pared alkali metal enolate reactant(24). The product (26), i. e. S-lower alkyl ether of2-thio-5-fluoroorotic acid lower alkyl ester or S-benzyl ether ofZ-thio-S-fluoroorotic acid lower alkyl ester, can be purified byconventional means, e. g. by recrystallization from inert solvents.

The third stage of the comprehensive embodiment of Process II compriseshydrolyzing the S-lower alkyl (or S- benzyl) ether of2-thio-5-fluoroorotic acid lower alkyl ester. The hydrolysis can beconveniently effected by conventional means, for example by heating thereactant (26) with an aqueous solution of a mineral acid, e. g. withconcentrated aqueous hydrochloric acid; alternatively, the reactant (26)can be saponified, e. g. by treating with an aqueous solution of astrong alkali such as aqueous KOH, followed by acid hydrolysis of theresulting solution of the alkali salt of S-lower alkyl (or S- benzyl)ether of 2-thio-5-fluoroorotic acid, e. g. by means of concentratedaqueous hydrochloric or hydrobrornic acid. The S-fluoroorotic acid (27)obtained as product can be purified by conventional means, for exampleby recrystallization from water or from an inert organic solvent.

The last stage of the comprehensive embodiment of Process II referred toabove comprises decarboxylating 5- fluoroorotic acid (27) at its meltingpoint, carbon dioxide being thereby liberated, and leaving a residue of5-fluorouracil. For a larger scale preparation, it is preferable to heatthe reactant (27), above its melting point, while dissolved in a highboiling inert solvent such as diphenyl, diphenyl oxide or mixturesthereof, until the evolution of carbon dioxide has ceased.

One preferred embodiment of the overall Process II shown in the flowsheet comprises condensing ethyl fluoroacetate with diethyl oxalate inthe presence of potassium ethoxide, thereby producing potassium enolateof diethyl fluorooxalacetate; condensing the latter withS-ethylisothiourea, thereby producing S-ethyl ether of Z-thiO-S-fluoroorotic acid ethyl ester; refluxing the latter with con :centratedaqueous hydrochloric acid thereby producing S-fiuoroorotic acid; andheating the latter above its melting point, while dissolved in an inertsolvent, thereby producing S-fluorouracil. In an alternative preferredembodiment, S-methylisothiourea is used in lieu of S-ethylisothiourea,S-rnethyl ether of Z-thio-S-luoroorotic acid ethyl ester being therebyproduced in lieu of S-ethyl ether of 2--thio-5-fiuoroorotic acid ethylester.

PROCESS III 7 As will be appreciated from the foregoing flow sheet forProcess III, a comprehensive embodiment of this 7 fluorouracil (32); andoxidizing the latter, thereby producing S-fluorouracil (33).

The first stage .of the comprehensive embodiment of Process III referredto above comprises splitting ofi the hydrocarbon radical attached to themercapto sulfur atom of the compound (31). This scission can be effectedby heating (31) with an anhydrous hydrogen halide. Preferably, anhydroushydrogen iodide is employed. An alternative method comprises heatingS-benzyl ether of 2- thio-S-fluorouracil with anhydrous aluminumbromide.

The second stage of the comprehensive embodiment of Process III referredto above comprises oxidizing the 2- thio-S-fluorouracil intermediateproduct (32). A suitable method of oxidation comprises heating the2-thio-5-fluorouracil with hydrogen peroxide.

One preferred embodiment of the overall Process III shown in the flowsheet comprises heating S-lower alkyl ether of 2-thio-5-fluorouraci1with anhydrous hydrogen iodide, thereby producing 2-thio-5-fluorouracil;and heating the latter with a concentrated aqueous solution of hydrogenperoxide, thereby producing S-fluorouracil.

PROCESS IV As will be appreciated from the foregoing flow sheet forProcess IV, a comprehensive embodiment of this fourth aspect of theinvention provides a process which comprises reacting a member (41) ofthe group consisting of S-lower alkyl ether of 2-thio-5-fiuorouracil andS-benzyl ether of Z-thio-S-fluorouracil with a phosphorus pentahalideselected from the group consisting of phosphorus pentachloride andphosphorus pentabromide, thereby producing the corresponding member (42)selected from the group consisting of 2-(loweralkyl)mercapto-4-chloro-5-fiuoropyrimidine, 2 (lower alkyl)mercapto 4bromo-5fluoropyrimidine, 2-benzylmercapto-4- chloro-S-fiuoropyrimidineand 2-benzylmercapto-4-bromo- S-fluoropyrimidine; aminating saidcorresponding member, thereby producing the corresponding member (43) ofthe group consisting of 2-(lower alkyl)mercapto-4-amino-S-fluoropyrimidine and 2-benzylmercapto-4-amino-S-fiuoropyrimidine (42) obtained as the product of this stage can bepurified by extracting with an inert solvent, such as diethyl ether.

The second stage of the comprehensive embodiment of Process IV referredto above comprises exchanging the 4-halo substituent in the intermediateproduct (42) for an amino group. This reaction can be effectedconveniently by reacting the product (42) with liquid ammonia under heatand pressure. Alternatively, alcoholic ammonia can be employed. Theresulting 2-(lower alkyl) mercapto (or 2-benzylmercapto-)4-amino-5-fluoropyrimidine (43) can be purified by recrystallizaton froman inert solvent.

The third stage of the comprehensive embodiment of Process IV referredto above comprises hydrolyzing the intermediate product (43). venientlyeffected by conventional means, for example by heating the reactant (43)with an aqueous solution of a mineral acid, e. g. with concentratedaqueous hydrobromic acid. The resulting product, S-fiuorocytosine (44),can be purified, for example, by recrystallization from water.

The hydrolysis can be con- H The last stage of the comprehensiveembodiment of and pressure, thereby producing S-ethylmercapto-4-aminoS-fiuoropyrimidine; refluxing the latter with concentrated aqueoushydrobromic acid, thereby producing S-fluorocytosine; and reacting thelatter with nitrous acid, thereby producing S-fluorouracil.

PROCESS V As will be appreciated from the foregoing flow sheet forProcess V, a comprehensive embodiment of this aspect of the inventionprovides a process which comprises condensing alkali metal enolate oflower alkyl fluoromalonaldehydate (51) under anhydrous conditions with amember (52) selected from the group consisting of 2- loweralkyl-pseudourea and Z-benzyl-pseudourea, thereby producing thecorresponding member (53) of the group consisting of 2-lower alkyl etherof S-fluorouracil and 2- benzyl ether of S-fluorouracil; and hydrolyzingsaid corresponding member (53), thereby producing 5-fluorouracil (54).

The first stage of the comprehensive embodiment of Process V referred toabove comprises condensing the alkali metal enolate of lower alkylfluoromalonaldehydrate (51) with (52), i. e. 2-lower alkyl-pseudourea or2-benzyl-pseudourea, under anhydrous conditions. Preferably, thereaction is effected by heating the reactants together in an anhydrousinert solvent such as methanol or ethanol, until the condensationreaction has been completed. It is recommended to use the alkali metalenolate reactant (51) as soon as possible after it has been prepared.The 2-lower alkyl-pseudourea or 2-benzyl-pseudourea is advantageouslyemployed in the form of its acid addition salt with a mineral acid, e.g. 2-methyl-pseudourea hydrochloride or 2-benzyl-pseudoureahydrochloride, in the presence of at least the equivalent amount ofalkali necessary to neutralize the mineral acid. The product (53) ofthis stage, 2-lower alkyl (or 2-benzyl) ether of 5- fluorouracil, can bepurified by conventional means, e. g. by recrystallization from inertsolvents.

The second stage of the comprehensive embodiment of Process V shown inthe flow sheet comprises hydrolyszing the ether (53), thereby producingS-fiuorouracil 4). conventional means, for example by heating thereactant (53) with an aqueous solution of a mineral acid, e. g. withconcentrated aqueous hydrochloric acid. The 5- fluorouracil obtained asproduct can be purified by conventional means, for example byrecrystallization from inert solvents or by sublimation in vacuo.

One preferred embodiment of the overall Process V shown in the flowsheet comprises condensing freshly prepared potassium enolate of ethylfluoromalonaldehydate under anhydrous conditions with2-n1ethyl-pseudourea, thereby producing 2-methyl ether ofS-fiuorouracil; and hydrolyzing the latter, thereby producingS-fiuorouracil.

It will be appreciated that the compounds represented in the foregoingflow sheets by the formulas (18), (19), (26), (27), (32), (44), and(53); and sometimes referred to in this specification, respectively, asS-lower alkyl ether of 2-thio-5-fluorouracil (or species thereof),andS-benzyl ether of 2-thio-5-fluorouracil, S-fluorouracil,

The hydrolysis can be conveniently etfected by can exist in tautomericforms, resulting from the shifting of a proton between a nitrogen atomand an oxygen atom. The invention includes all of the tautomeric formsof said compounds.

The compounds sometimes referred to herein as S-lower alkyl ether ofZ-thio-S-Iluorouracil (or species thereof), and S-benzyl ether of2-thio-5fluorouracil,

S-fiuoroorotic acid,

2-thio-5-iuorouracil, and

2-lower alkyl ether of 5-fluorouracil (or and 2-benzyl ether ofS-fluorouracil species thereof),

exhibit acidic properties, and form salts with bases. The inventionincludes salts obtained by reacting said compounds, respectively, withmedicinally acceptable bases, e. g. alkali metal hydroxides, alkalineearth metal hydroxides, ammonia, non-toxic organic bases such asethanolarnine, and the like.

The compounds sometimes referred to herein as 2-(loweralkyl)mercapto-4-amino-S-fiuoropyrimidine (or species thereof) and2-benzyhnercapto-4-amino-S-fiuoropyrimidine, and

S-fiuorocytosine;

exhibit basic properties, and form acid addition salts. The inventionincludes acid addition salts formed by reacting said compounds withmedicinally acceptable acids, e. g. mineral acids, such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid;non-toxic organic acids, such as ethanesulfonic acid, toluenesulfonicacid, tartaric acid, citric acid; and the like.

The novel compound S-fluorouracil and its salts with medicinallyacceptable bases are useful as germicidal agents, being active, forexample, against gram-positive and gram-negative and other bacteria,such as Proteus vulgaris, Staphylococcus aureus, Escherichia coli,Pseudomonas aeruginosa, Bacillus subtilis, Bacillus megatherium, Sarcinalutea, Corynebacterium simplex and the like; against protozoa such as Tetrahymena geleii and the like; against fungi such as Scopulariopsisbrevicaulis and the like. They are also useful as antimetabolites, beingactive to inhibit the growth of Lactobacillus arabinosus, Lactobacillusleichmannii, Streptococcus faecalis, Saccharomyces carlsbergensis andthe like, e. g. by interfering with the utilization of uracil, thymine,cytosine and thymidine by the microorganism. For example, S-fluorouracilcan be applied locally, in solution in a physiologically inert liquidmedium, e. g. saline solution, to lesions infected with Pseudomonasaeruginosa, as an antibacterial agent to terminate the development ofthe infection.

The invention is further disclosed in the following examples, which areillustrative but not limitative thereof. Temperatures are stated indegrees centigrade, corrected.

Example 1 A mixture of 200 g. (2 mols) of dry sodium fluoroaeetate and442 g. (2.86 mols) of diethyl sulfate was refluxed for three andone-half hours in an oil bath. The reaction mixture was then distilledthrough a fractionating column, yielding 177.3 g. of crude ethylfluoroacetate, having a boiling range of 116-l20. The material wasredistilled through a fractionating column, yielding purified ethylfluoroacetate boiling at 114118.

Example 2 i In a 2-liter, 3-neck, round bottom flask, provided withstirrer, dropping funnel and reflux condenser, was placed 880 ml. ofabsolute diethyl ether, and 47.6 g. (1.22 mols) of potassium, cut into 5mm. pieces, was suspended therein. 220 ml. of absolute ethanol was addeddropwise, while stirring, whereby the heat of reaction producedrefluxing. In order to obtain complete dissolution of the potassium, themixture was finally refluxed on a steam bath. The reaction mixture wasthen cooled in an ice bath, and a mixture of g. (1.22 mols) of ethylfluoroacetate and 96.4 g. (1.3 mols) of freshly distilled ethyl formatewas added dropwise, while stirring and cooling, over a period of two andone-half hours. Upon completion of the addition of the ethyl formate,the reaction mixture was stirred for an additional hour while cooling,and then was allowed to stand overnight at room temperature. At the endof this time the crystalline precipitate which had formed was filteredoff with suction, washed with diethyl ether, and dried in a vacuumdesiccator. The product comprised essentially the potassium enolate ofethyl fluoromalonaldehydate (alternative nomenclature, the potassiumsalt of fluoromalonaldehydic acid ethyl ester).

Example 3 A mixture of 103.6 g. (0.6 mol) of the freshly preparedpotassium enolate of ethyl fluoromalonaldehydate, 83.4 g. (0.3 mol) ofS-methylisothiouronium sulfate and 32.5 g. (0.6 mol) of sodium methoxidewas refluxed with stirring in 1500 ml. of absolute methanol. At firstthe reactants dissolved to a great extent, but very shortly thereafterprecipitation occurred. The reaction mixture was refluxed for two hoursand at the end of this time was evaporated to dryness in vacuo. Theresidue was treated with 280 ml. of water; incomplete dissolution wasobserved. The mixture obtained was clarified by filtering it throughcharcoal. The filtrate was acidified (to a slight Congo acid reaction)by adding concentrated aqueous hydrochloric acid, containing 37% byweight HCl (48 ml. required). The material which crystallized from theacidified solution was filtered off, washed free of sulfates with waterand dried at 100, yielding crude S-methyl ether of 2-thio-5fluorouracil,having a melting range from 202 to 221. The latter material wasrecrystallized by dissolving it in 2035 ml. of boiling ethyl acetate andcooling to minus 20, yielding S-methyl ether of 2-thio-5- fluorouracil,M. P. 230-237, in a sufficient state of purity that it could be useddirectly for the next step. A sample of the material was recrystallizedfrom water (alternatively, from ethyl acetate) thereby raising themelting point to 24l243. For analysis the material was further purifiedby subliming it in vacuo at /0.1 mm.

The S-methyl ether of 2-thio-5-tluorouracil so obtained was an acidicmaterial, which formed salts upon reaction with bases. For example, itdissolved, with salt formation, in dilute aqueous sodium hydroxidesolution, in aqueous ammonia, and in an aqueous solution ofethauolamine.

Example 4 A solution of 10.0 g. of purified S-methyl ether of 2-thio-S-fluorouracil, M. P. 230237, in 150 ml. of concentrated aqueoushydrochloric acid (containing approximately 37% by weight HCl) wasrefluxed under nitrogen for four hours. The reaction mixture was thenevaporated in vacuo. The crystalline brownish residue was recrystallizedfrom water. The resulting recrystallized product was further purified bysublimation in vacuo at -200 (bath temperature)/O.1 mm. pressure. Therewas obtained 5-fiuorouracil, in the form of colorless or pinkishtancrystals, M. P. 282-283 (with decomposition).

The S-fluorouracil so obtained was an acidic material which formed saltsupon neutralization with bases. It dissolved, with salt formation, indilute aqueous potassium hydroxide solution, in aqueous ammonia and inaqueous ethanolamine.

Example A mixture of 17.3 g. (0.1 mol) of the freshly prepared potassiumenolate of ethyl fluoromalonaldehydate, 18.5 g. (0.1 mol) ofS-ethylisothiouronium bromide and a solution of 2.3 g. (0.1 mol) ofsodium in 250 m1. of absolute ethanol was refluxed for two hours whilestirring. The reaction mixture, consisting of a slightly yellowishcrystalline material suspended in the liquid, was evaporated to drynessin vacuo. The residue was dissolved in ml. of warm water and thesolution was clarified by filtering through charcoal. The filtrate wascooled in an ice bath and acidified (to a slight Congo acid reaction) byadding concentrated aqueous hydrochloric acid (5.8 ml. required). Thematerial which crystallized from the acidified solution was filteredofi, washed free of bromides with ice cold water and dried at yieldingcrude S-ethyl ether of 2-thio-5-fluorouracil, having a melting rangefrom 164 to The latter material was suspended in a 1:4 (by volume)mixture of toluene and ligroin (boiling point 90120) and refluxed for afew minutes. Partial solution was obtained. The insoluble material wasfiltered from the boiling mixture and suspended in toluene. Thesuspension was brought to boiling and filtered hot. Upon cooling in anice bath, the filtrate deposited crystals which were filtered off anddried at 100. There was thus obtained purified S-ethyl ether of2-thio-5-fluorouracil melting from 181 to (alternative nomenclatures:2-ethylmercapto-4-hydroxy-5-fluoropyrimidine, orZ-ethylmercapto-5-fluoro-4(3H)-pyrimidinone). The material was againrecrystallized from ethyl acetate, yielding further purified compoundmelting at l91. This material was sufficiently pure to be used for thenext step. For analysis a sample of 300 mg. was again recrystallizedfrom 10 ml. of ethyl acetate to yield 210 mg. of pure compound meltingat 192-l93. The S-ethyl ether of 2-thio-5-fluorouracil thus obtained wasan acidic material, which formed salts upon reaction with bases. Forexample, it dissolved, with salt formation, in dilute aqueous sodiumhydroxide solution and in aqueous ammonia.

Example 6 A solution of 0.52 g. of purified S-ethyl ether of 2-thio-S-fluorouracil, M. P. 190-191, in 10 ml. of concentrated aqueoushydrochloric acid (37% by weight HCl) was refluxed for four and one-halfhours. The reaction mixture was then evaporated in vacuo. Thecrystalline tan residue was recrystallized from 4 ml. of water.. Thecrystals obtained were washed with cold water and then with methanol,yielding 5-fluorouracil, M. P. 281282 (with decomposition). Sublimationin vacuo (0.1 mm., 190 bath temperature) raised the melting point to282- 283.

Example 7 A solution of 43 g. (0.25 mol) of freshly prepared ethylpotassio-fluoromalonaldehydate, 50.6 g. (0.25 mol) ofS-benzylisothiouronium chloride and 13.5 g. (0.25 mol) of sodiummethoxide in 640 ml. of methanol was refluxed for two hours whilestirring. The reaction mixture was evaporated to dryness in vacuo, andthe residue was taken up in 220 ml. of water. The resulting turbidsolution was made alkaline to phenolphthalein by addition of 12 ml. of 2N aqueous sodium hydroxide solution and was then extracted with threeportions of diethyl ether, each portion consisting of 60 ml. The aqueouslayer was then acidified with 16 ml. of concentrated aqueoushydrochloric acid and cooled with ice. The precipitated crystals werefiltered and washed chlorine-free with water and then with diethylether. There was thus obtained crude S-benzyl ether of2-thio-5-fluorouracil (alternative nomenclature:2-benzylrnercapto-5-fluoro-4 3H) -pyrimidinone) having melting point180-189. Upon recrystallization 12 from ethanol, the melting point wasraised to 205-206.- A second recrystallization from ethanol raised themelting point to 216-218.

Example 8 A potassium ethoxide solution in toluene was prepared from47.6 g. (1.214 mols) of potassium, 880 ml. of toluene and 190 ml. ofethanol, 300 ml. of toluene-ethanol mixture being distilled off aftercomplete dissolution of the potassium. To the ice-cold solution, fromwhich potassium ethoxide began to crystallize, there was added, undernitrogen, 355 g. (328 ml., 2.428 mols) of diethyl oxalate. A clearyellow solution resulted. While cooling and stirring, 135 g. (1.214mols) of ethyl fluoroacetate was added dropwise during one and one-halfhours. Stirring and cooling were continued for one hour, then thecrystallizing mixture was allowed to stand at room temperatureovernight. The readily settling colorless crystals were filtered, washedwith diethyl ether and dried in vacuo, yielding potassium enolate ofdiethyl fluorooxalacetate (alternative nomenclature, diethylpotassio-fluorooaxalacetate).

Example 9 Under nitrogen, to a solution of 6.8 g. (0.295-mol) of sodiumin 750 ml. of ethanol there was added 72.5 g. (0.295 mol) of thepotassium enolate of diethyl fiuorooxalacetate and 54.6 g. (0.295 mol)of S-ethylisothiouronium bromide. Almost complete dissolution wasobserved, followed by crystallization. T he mixture was refluxed undernitrogen while stirring for two hours, and then was evaporated todryness in vacuo. The residue was dissolved in 200 ml. of ice-coldwater, the solution obtained was clarified by filtration throughcharcoal and extraction of some oil by two portions of diethyl ether,each containing 50 ml. The aqueous layer was cooled in ice and acidifiedto pH 2 by addition of 50 ml. of dilute aqueous hydrochloric acid (19%by weight HCl). The crystallized material was filtered, washedchlorine-free with water and dried at 100, yielding Sethyl ether of2-thio-5-fluoroorotic acid ethyl ester (alternative nomenclature:Z-ethylmercapto-4-hydroxy-S-fluoro--pyrimidinecarboxylic acid ethylester) M. P. 166167. Recrystallization of the product from toluene whilecooling to -20 yielded purer material having M. P. l67168. For analysis,0.74 g. of the latter material was again recrystallized from 10 ml. oftoluene to yield 0.7 g., M. P. 168l69.

Example 10 Under nitrogen, 4 g. (0.0162 mol) of S-ethyl ether of2-thio-5-fluoroorotic acid ethyl ester, M. P. 166-167, was refluxed in70 ml. of concentrated aqueous hydrochloric acid (containing 37% byweight HCl) for four hours. The mixture was cooled in ice and thecrystallized acid was filtered, washed chlorine-free with water anddried at 100. There was thus obtained S-fluoroorotic acid monohydrate.The compound retained its water of crystallization very tenaciously. Theacid melted with decarboxylation at 255; the residue, which was 5-fluorouracil, solidified and then melted at 278279. For analysis, asample of S-fluoroorotic acid monohydrate was recrystallized from ca. 35volumes of water.

A samle of 5-fluoroorotic acid monohydrate was sublimed in vacuo at260270 (bath temperature) and 0.1

d mm., yielding the anhydrous acid, which melted with decarboxylationand resolidification like the monohydrate.

The S-fluoroorotic acid (in both the anhydrous and the monohydrateforms) thus obtained was an acidic mate rial. Upon reaction with bases,it formed salts. Thus, it dissolved in aqueous sodium hydroxide andpotassium hydroxide solutions, forming sodium and potassium salts,respectively.

S-fluoroorotic acid is useful as an antimetabolite, being active toinhibit the growth of L. leichmannii, L. Cassi and S. faecalis, forexample. It is also active as a germicidal agent, against Pseazlomonasaeruginosa and similar gram-negative organisms.

Example 11 Example 12 A mixture of 100 g. (0.41 mol) of diethylpotassiofiuorooxalacetate, 57.2 g. (0.21 mol) of S-methylisothiouroniumsulfate and a solution of 44.3 g. (0.82 mol) of sodium methoxide in 1100ml. of methanol was refluxed under nitrogen and worked up as describedin Example 9 above for the ethylmercapto ester. In this case,transesterification took place and S-methyl ether of 2-thio-5-fiuoroorotic acid methyl ester was obtained; M. P. 195 197. Foranalysis, a sample was recrystallized from 60 volumes of toluene: M. P.199 201 Example 13 A solution of 1.84 g. (0.08 mol) of sodium in 150 ml.of ethanol, 8.25 g. (0.04 mol) of diethyl potassio-fluorooxalacetate and5.57 g. (0.02 mol) of S-methylisothiouronium sulfate was refluxed fortwo hours and worked up as described in Example 9 above, yieldingS-methyl ether of Z-thio-S-fiuoroorotic acid ethyl ester, M. P. 177 178.For analysis, the compound was recrystallized from 25 volumes oftoluene: M. P. 183l84.

Example 14 T o a solution of g. (0.031 mol) of S-methyl ether of 2-thio-S-fiuorouracil in 128 ml. of acetic acid and 32 ml. of aceticanhydride, which was stirred and refluxed, was added dropwise a mixtureof 8 ml. (0.106 mol) of 55% hydriodic acid (d=1.7), 120 ml. of aceticacid and 40 ml. of acetic anhydride. Refluxing was continued for an hourafter all the hydriodic acid had been added. The reaction mixture wasthen evaporated in vacuo to a volume of ml., and the concentrate wascooled in ice. The resulting crystalline precipitate was filtered off,washed with a mixture of acetic acid and petroleum ether and finallywith petroleum ether. There was thus obtained 2-thio-5-fiuorouracil, M.P. 219-221. Recrystallization from water raised the melting point to222224, and a second recrystallization from water raised the meltingpoint to 225-226.

9.5 g. of S-ethyl ether of 2-thio-5-fiuorouracil was refluxed withhydriodic acid, in the same manner indicated above except that therefluxing time was extended to two hours. There was thus obtained2-thio-5-fiuorouracil.

Example 15 To a solution of 0.614 g. (0.0042 mol) of 2-thio-5-fluorouracil in 19 ml. of water was added 3 ml. of hydrogen peroxide.The mixture was cooled in ice, and 3 ml. of 5 N sodium hydroxide wasadded slowly. The reaction mixture was allowed to warm up to roomtemperature slowly and then was refluxed for one and onehalf hours. Itwas then concentrated in vacuo to a volume of 4 ml, cooled, andacidified with 0.5 ml. of concentrated hydrochloric acid. The acidifiedreaction mixture was evaporated to dryness and the crystalline residuewas extracted with four portions of hot methanol, each portionconsisting of 5 ml. The combined methanolic extracts were evaporated andthe residue was dissolved in 0.5 ml. of hot water. Upon cooling, crudeS-fluorouracil (M. P. 258) crystallized. Recrystallization raised themelting point to 274. Admixture of the recrystallized i the mixture for30 minutes, the oil became crystalline.

14 product with an authentic sample of S-fluorouraoil resulted in nodepression of the melting point.

Example 1 6 To a solution of 4.3 g. (0.016 mol) of anhydrous alu minumbromide in 28 ml. of dry toluene was added, While stirring, 3.65 g.(0.0154 mol) of S-benzyl ether of 2-thio- 5-fiuorouracil, M. P.2'l6-218. The mixture was heated to 60 for six hours while stirringcontinuously, then was cooled to 25. 5 ml. of water was added, resultingin the formation of an oily layer; but upon stirring The crystals werefiltered off and washed with cold water, then dissolved in 30 ml. of hotN hydrochlori acid. The solution was cooled to room temperature, and wasextracted with three portions of butanol, each consisting of 16 ml. Thecombined butanol extracts were evaporated, leaving a residue of.Z-thio-S-fluorouracil, M. P. 209- 211. TWO recrystallizations from waterraised the melting point to 221223.

Example 17 A mixture of 10 g. of S-ethyl ether of 2-thio-5-fiuorouraciland 12 g. of phosphorus pentachloride was heated on a steam bath untilthe mixture was liquefied to a clear solution. The phosphorusoxychloride formed Was removed by heating in vacuo on a steam bath. Tothe oily residue was added crushed ice, whereupon crystallization wasobserved. The reaction mixture was extracted with three portions ofdiethyl ether, each consisting of 25 ml.; and the ethereal solution wasdried over sodium sulfate. The dried solution upon evaporation yieldedan oil comprising essentially Z-ethylmercapto 4 chloro 5fluoropyrimidine, which crystallized only below room temperature.

Example 18 The oil obtained in Example 17 above was autoclaved for 12hours with 120 ml. of liquid ammonia, in a boiling water bath. Theammonia was then evaporated, and the semisolid reaction product wastaken up with 100 ml. of water and 10 ml. of ethanol. The crystals whichseparated were filtered and washed chlorine-free with water, leaving aresidue of 2-ethylmercapto-4-amino-5- flnoropyrimidine, M. P. 94-95. Asample was recrystallized from 30 volumes of ligroin (B. P. 90 -120) therecrystallized product melted at 94 95.

The 2-ethylmercapto-4-amino-5-fluoropyrimidine, obtained as describedabove, was a basic material, which formed acid addition salts uponreaction with acids. Thus, it dissolved in dilute aqueous hydrochloricacid, thereby forming the hydrochloride salt.

Example 19 8.53 g. of crude 2-ethyhnercapto-4-amino-5-fiuoropyrimidine,M. P. 9091, was refluxed for four hours with ml. of concentrated aqueoushydrobromic acid (containing 48% by weight HBr) in a nitrogenatmosphere. The solution was evaporated in vacuo, and the residue wastwice taken up with water and re-evaporated. The final residue wasdissolved in 25 m1. of hot water and the solution was clarified byfiltration through charcoal. Upon addition of 11 ml. of concentratedammonia, the base precipitated. After cooling in ice, it was filteredand washed with cold water and ethanol, yielding S-fluorocytosine(alternative nomenclature: 2-hydroxy-4-arnino S-fiuoropyrimidine). Theproduct melted with decomposition at approximately 297.

S-fluorocytosine is a basic material, and forms acid addition salts uponreaction with acids. Thus, it dissolved in dilute aqueous hydrochloricacid and dilute aqueous hydrobromic acid, with formation ofS-fiuorocytosine hydrochloride and S-fiuorocytosine hydrobromide,respectively.

S-fiuorocytosine and its salts are useful as antimeta- 15 bolites,-being active, for example, to inhibit the growth of L. leichmannii, Lcasei, S. faecalz's, and the like.

Example 20 To a solution of 0.26 g. (0.002 mol) of S-fluorocytosine inml. of water and 5 ml. of acetic acid there was added a solution of 0.69g. (0.01 mol) of sodium nitrite in ml. of water in three portions over aperiod of one hour. The mixture was allowed to stand overnight, and thenwas evaporated on a steam bath. The semi-solid residue obtained wasdissolved in 10 ml. of Water, the solution was rendered alkaline byaddition of 3 ml. of 3 N sodium hydroxide and then was passed through a1.8 cm. x 20 cm. column of Dowex 1X4 (Dow Chemical Co., Midland,Michigan: an anion exchange resin consisting of a crosslinked copolymerof styrene with divinyl benzene [4% of the latter], containingquaternary ammonium groups as the functional groups), 100-200 mesh size,previously saturated with formate ion by washing with 0.1 N aqueousformic acid. After washing the column with 250 ml. of water, elution wasperformed with 0.1 N aqueous formic acid, 50 ml. fractions being takenand examined for ultraviolet absorption at 265 III/1.. Only fraction No.5 contained substantial amounts of absorbing material. This fractionupon evaporation to dryness yielded S-fluorouracil, which was identifiedby mixed melting point and comparison of the ultraviolet spectrum atpH=1 and 7.2 with an authentic specimen.

Example 21 A solution of 11 g. (0.0638 mol) of potassium enolate ofethyl fiuoromalonaldehydrate, 7.05 g. (0.0638 mol) of Z-methylpseudoureahydrochloride and 3.78 g. (0.07 mol) of sodium methoxide in 200 ml. ofmethanol was refluxed with stirring for two hours. The resulting cloudyliquid was evaporated to dryness in vacuo on the Water bath, the residueobtained was taken up with 18 ml. of water, the solution was filteredfrom undissolved material, cooled in ice and acidified to congo paperwith 2.2 ml. of concentrated hydrochloric acid. The crystallineprecipitate was filtered and washed with water and then with diethylether. There was thus obtained the 2- methyl ether of S-fluorouracil(alternative momenclature, 2 methoxy 5 fluoro 4(3H) pyrimidinone); M. P.190-l92. The crude material was recrystallized from water: M. P. 205206. A second recrystallization raised the melting point to 206-207. Thesubstance was sublimed at 0.1 mm. and 155 l60 bath temperature, butthere was no change in the melting point.

Example 22 Fifty mg. of 2-methoxy-5-fluoro-4(3H)-pyrimidinone, M. P.206-207, was refluxed for four hours with 5 ml. of concentrated aqueoushydrochloric acid (37% by weight HQ) in a nitrogen atmosphere. Thesolution was evaporated to dryness, taken up with water andreevaporated, yielding 5-fiuorouracil, M. P. 282283. The compound wasfurther identified by mixed melting point with an authentic specimen.

Example 23 In a 3 liter flask provided with stirrer, reflux condenser,dropping funnel, thermometer and gas inlet tube by which a continuousstream of nitrogen was provided during all operations, was placed 880ml. of toluene and then 47.6 g. (1.214 mol) of potassium. The flask washeated in an oil bath to ca. 80 until the potassium melted. The metalwas finely divided by stirring. Then 190 ml. of ethanol was dropped inat such a rate (ca. 30 minutes required) that slight refluxing resulted.Heating was continued until all potassium was dissolved (additional 45minutes required). Then ca. 250 ml. of tolueneethanol mixture wasdistilled oil. Upon cooling in an ice bath. some crystallization ofpotassium ethoxide was observed. 146 g. (2.428 mol, 149 ml.) of drymethyl formate (distilled over CaCO3) was added, whereupon a clearcolorless solution resulted. With continuous cooling in an ice bath andstirring, g. (1.214 mol) of ethyl fluoroacetate was added dropwisewithin two hours. After one hour of further stirring, the ice bath wasremoved and the crystallizing mixture was allowed to stand at roomtemperature overnight. The almost colorless crystals were filtered,Washed with ether and dried in vacuo at room temperature, yieldingpotassium enolate of ethyl fiuoromalonaldehydate (alternativenomenclature, potassio ethyl formylfluoroacetate).

Example 24 To a solution of 6.7 g. (0.29 mol) of sodium in 720 ml. ofabsolute ethanol, prepared in a 2-liter, 3-neck flask provided withstirrer and reflux condenser, was added 50 g. (0.29 mol) of the abovepotassium enolate (from Example 23) and 53.6 g. (0.29 mol) of ethylisothiouronium bromide. An almost complete solution resulted which soonbecame cloudy by crystallization. The mixture was refluxed withcontinuous stirring on a water bath for two hours. The alcohol wasdistilled off in vacuo; the residue obtained was dissolved in ml. ofwater, the solution was clarified by filtering through charcoal, cooledin an ice bath and acidified (to Congo paper) by addition of 22 ml. ofconcentrated hydrochloric acid. The crystalline precipitate wasfiltered, washed chlorine free with water and dried at 100, yielding2-ethylmercapto-5-fluoro-4(3H)-pyrimidinone, M. P. 179. The crudematerial was dissolved in 710 ml. of boiling ethyl acetate, the hotsolution was filtered and cooled to l0. The crystals were filtered andwashed with cold ethyl acetate. The melting point was thus raised to189l90.

We claim:

. 1. A compound selected from the group consisting of 5-fluorouracil andsalts thereof.

3. A process of making S-fluorouracil which comprises hydrolyzing amember of the group consisting of S-lower alkyl ether of2-thio-5-fluorouracil and S-benzyl ether of 2-thio-5-fiuorouracil.

4. A process of making S-lower alkyl ether of 2-thio- 5-fluorouracilwhich comprises condensing freshly prepared alkali metal enolate oflower alkyl fluoromalonaldehydrate under anhydrous conditions withS-(lower alkyl) isothiourea.

5. A process of making S-benzyl ether of 2-thio-5 fluorouracil whichcomprises condensing freshly preparen alkali metal enolate of loweralkyl fluoromalonaldehydate under anhydrous conditions withS-benzylisothio'urea.

6. A process of making S-lower alkyl ether of 2-thio S-fluorouracilwhich comprises condensing freshly prepared potassium enolate of loweralkyl fluoromalonalde hydate under anhydrous condition with S-(loweralkyl) isothiourea.

7. A process according to claim 6 wherein freshly prepared potassiumenolate of ethyl fluoromalonaldehydate is employed.

8. A process of making S-fluorouracil which comprises decarboxylatingS-fluoroorotic acid.

9. A process which comprises splitting oi the S-hy drocarbon substituentfrom a member selected from the group consisting of S-lower alkyl etherof 2thio-5- fluorouracil and S-benzyl ether of 2-thio-5-fiuorouracil,thereby producing 2-thio-5-fluorouracil; and oxidizing the latter,thereby producing 5-luorouracil.

10. A process of making S-iluorouracil which comprises oxidizing2-thio-5-fluorouracil.

11. A process of making 5-fluorouraci1 which comprises reacting5fluorocytosine with nitrous acid.

12. A process of making 5-luorouracil which comprises hydrolyzing amember of the group consisting of 7 8 2-l0wer alkyl ether ofS-fluorouracil and 2-benzyl ether 2,666,764 Lanzillotti et al. Jan. 19,1954 of S-fluorouracil. 2,753,347 Miller July 3, 1956 2,753,348 MillerJuly 3, 1956 References Cited in the file of this patent r OTHERREFERENCES UNITED STATES PATENTS a Johnson: Amer. Chem. Journal, vol.40, pp. l9-36 2,138,756 Boese Nov. 29, 1938 (1908). 2,405,820 Faith Aug.13, 1946 Johnson: Jour. Amer. Chem. Soc., 65 1218-20 (1943). 2,585,615Barrett Feb. 12, 1952 Hilbert et al.: Jour. Amer. Chem. Soc., 56 13492,609,372 Ziegler Sept. 2, 1952 10 (1934).

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF 5-FLUOROURACIL ANDSALTS THEREOF.